Method of screening remedy for heart disease and medicinal composition for treating heart disease

ABSTRACT

A method for screening a substance that inhibits the onset of anti-cardiac troponin I autoantibody-related disease, a pharmaceutical composition and a base material for therapy of cardiac disease that contains the substance obtained by aforesaid method thereof, a therapeutic apparatus that removes anti-troponin I autoantibody for aforesaid antibody related disease, a method of making an animal model for evaluating cardiac disease characterized by administrating anti-cardiac troponin I antibody, a method of selection of a therapeutic substance for cardiac disease characterized by using aforesaid animals, and a diagnosis of dilated cardiomyopathy characterized by measuring anti-cardiac troponin I autoantibody. An apparatus of the present invention that removes anti-cardiac troponin I antibody and a pharmaceutical composition for therapy of the antibody related disease may be useful for a therapy and/or prevention of cardiac disease.

TECHNICAL FIELD

The present invention relates to; a screening method of a substance thatinhibits the onset of anti-troponin I autoantibody-related disease; apharmaceutical composition or a base material for therapy of cardiacdisease including a substance obtained by the above method; atherapeutic apparatus that removes anti-troponin I autoantibodies foraforesaid antibody related disease; a method of making an animal modelfor evaluating cardiac disease characterized by administratinganti-cardiac troponin I antibodies; a method of screening a substancefor therapy of cardiac disease characterized by using the animal; and adiagnosis of dilated cardiomyopathy characterized by measuring theamount of anti-cardiac troponin I autoantibodies.

BACKGROUND ART

Dilated cardiomyopathy is one of the myocardial diseases characterizedby progressive depression of myocardial contractile function and dilatedventricle, and the dysfunction of depressing dilated left ventricle ispresent. This disease often develops chronically and the prognosis ispoor in the majority of cases. Therefore, in Europe and the UnitedStates, cardiac transplantation is necessary for this disease in manycases, and above 90% of cardiac transplantation cases in Japan werethose of patients with this disease. 30% of dilated cardiomyopathypatients are said to have a congenital mutation in the gene that codesan important component of myocardium, which links cytoskeleton to cellmatrix. However, the causes of the rest of cases are unknown. In bothcases, the onset rate is very high (36.5 in 100,000 people) and thedeath rate is also high. However, there is no effective therapy exceptfor cardiac transplantation.

In addition to ischemic, toxic, metabolic, infectious, and geneticcauses, autoimmunity has been suspected to be one of the main causes.Several papers have reported that the patients of myocarditis orcryptogenic dilated cardiomyopathy have heart reactive autoantibodies.Antibodies to the mitochondrial ADP/ATP translocator or βb01-adrenoceptor, which are often found in human patients, have beenshown to enhance Ca²⁺ current (Journal of Experimental Medicine, 1988,168(6), p 2105-2019, European Journal of Pharmacology, 2001, 423(2-3), p115-119). In addition, immunoabsorption has been reported to havebeneficial effects. (Circulation, 2001, 103(22), p 2681-2686). StephanB. Felix proved that the therapy with immunoabsorption made patients'hemodynamics improved and the column eluent, which contains harvestedantibodies from immunoabsorption column, decreased cell contraction inmyocardial cells because of calcium current suppression (Journal of theAmerican College of Cardiology, 2002, 39(4), p 646-652). Experimentalstudies in rodents have shown that cardiotropic viral infections orimmunization with cardiac antigens can elicit injury of cardiomyocytes,leading to cardiomyopathy with concomitant production of heart-reactiveautoantibodies and cytotoxic T cells (Circulation, 1982, 65(6), p1230-1235, Clinical Immunology and Immunopathology, 1987, 43, 1, p129-139, Journal of Molecular and Cellular Cardiology, 1997, 29(2), p641-655, Journal of Immunology, 1987, 139(11), p 3630-3636). However,because autoimmune responses observed in the rodent models and humanpatients could be secondary to heart inflammation, the involvement ofautoimmunity in the pathogenesis of dilated cardiomyopathy is stilldebatable (Journal of Immunology, 1990, 145(12), p 4094-4100).

PD-1 is an immune inhibitory receptor belonging to CD28/CTLA-4 familyand inhibits antigen receptor-mediated signaling by recruiting SHP-2upon engagement with its ligands, PD-L1 or PD-L2 (Current OpinionImmunology, 2002, 14(6), p 779-782). C57BL/B6 PD-1-deficient micedeveloped lupus-like glomerulonephritis and arthritis (Current OpinionImmunology, 2002, 14(6), p 779-782). The inventors have recently shownthat BALB/c PD-1-deficient mice develop dilated cardiomyopathy (Science,2001, 291(5502), p 319-322, WO02/39813).

The dilated heart of the mice showed prominent depositions of immunecomplex on myocardial cells. Furthermore, sera from PD-1-deficient micecontained high-titer autoantibodies against a heart-specific, 30-kDaprotein. Although these results support the hypothesis that autoimmunityis a probable cause of dilated cardiomyopathy, there was no directevidence. Therefore, it is essential to identify the 30-kDa antigen andassess the pathogenic role of the antigen-specific autoimmune reactionin dilated cardiomyopathy.

DISCLOSURE OF THE INVENTION

The task of the present invention is the provision of a pharmaceuticalcomposition and a therapeutic apparatus for anti-cardiac troponin Iautoantibody-related disease, and a diagnosis thereof.

The inventors of the present invention showed that anti-cardiac troponinI autoantibodies existed in serum of PD-1-deficient mouse, andidentified that autoantibodies against cardiac troponin I were the onsetfactor of dilated cardiomyopathy.

The inventors of the present invention thought that the anti-cardiactroponin I autoantibodies were involved in the onset of dilatedcardiomyopathy in PD-1-deficient mouse because of the existence ofanti-cardiac troponin I autoantibody in serum of PD-1-deficient mouseand the result of the analysis in the depositions of immune complex onthe surface of myocardial cells and the like. Concretely, binding ofanti-cardiac troponin I autoantibody to cardiac troponin I resulted incompromising cardiac function. Moreover, we showed thatvoltage-dependent calcium current in normal myocardial cells wasincreased by addition of monoclonal anti-cardiac troponin Iautoantibodies suggesting that cardiac troponin I expressed onmyocardial cells has a role in controlling calcium current. From aboveresults and discussion, the inventors of the present invention concludedthat the anti-cardiac troponin I caused dilated cardiomyopathy bychronic rise in calcium current of myocardial cells. Therefore, theanti-cardiac troponin I autoantibody-related disease are thought to betreatable by a removal of anti-cardiac troponin I autoantibodies, aninhibition of interaction between anti-cardiac troponin I autoantibodyand cardiac troponin I, an inhibition of effect of anti-cardiac troponinI autoantibody to its target tissues, and an inhibition of anti-cardiactroponin I autoantibody production. The diagnosis of the anti-cardiactroponin I autoantibody-related disease may be carried out by measuringthe amount of antibody in collected blood, sera, blood plasma, urine andtissue of the patients.

Based on above finding and discussion, the inventors completed theinvention related to the following; a screening method of a substancethat inhibits the onset of anti-cardiac troponin I autoantibody-relateddiseases, a pharmaceutical composition and a base material for therapyof cardiac disease that contains a substance obtained by the abovemethod, a therapeutic apparatus for anti-cardiac troponin Iautoantibody-related diseases by removing the antibody, a method ofmaking animal model for evaluating cardiac disease, characterized byadministration of anti-cardiac troponin I autoantibody, a method ofselection of a therapeutic substance for cardiac disease characterizedby use of the above animal and diagnostic method of dilatedcardiomyopathy.

The present invention relates to the followings:

1. A screening method of a substance that inhibits interaction betweenanti-troponin I antibody and cardiac troponin I, which comprisesmeasuring and evaluating the inhibitory activity of a test substance oninteraction between anti-cardiac troponin I antibody and cardiactroponin I after contacting the anti-cardiac troponin I antibody, thecardiac troponin I and the test substance.

2. A screening method of a substance that inhibits the effect ofanti-troponin I to a target tissue, which comprises measuring andevaluating the inhibitory activity of a test substance on the effect ofanti-cardiac troponin I antibody on a target tissue after contacting theanti-cardiac troponin I, the target tissue and the test substance.

3. The screening method according to the above 2, wherein the targettissue is a myocardial cell.

4. A pharmaceutical composition of therapy for cardiac disease which ismade by steps comprising a step of selecting a substance with activityby the screening method according to any one of the above 1 to 3, a stepof manufacturing the above selected substance, and a step of mixing thesubstance manufactured by the above described step with apharmaceutically acceptable solvent for formulation.

5. A pharmaceutical composition of therapy for cardiac disease, whichcomprises at least one selected from cardiac troponin I protein, apartial protein thereof, and a modified protein thereof as an activeingredient.

6. The pharmaceutical composition of therapy for cardiac diseaseaccording to the above 4 or 5, wherein the cardiac disease is dilatedcardiomyopathy.

7. A therapeutic base material for cardiac disease coupled with asubstance selected by the method according to any one of the above 1 to3.

8. A therapeutic base material for cardiac disease coupled with at leastone selected from cardiac troponin I, its partial protein, and amodified protein thereof

9. The therapeutic base material for cardiac disease according to theabove 7 or 8, wherein the cardiac disease is dilated cardiomyopathy.

10. An apparatus for therapy of cardiac disease, which comprises aplasma separating apparatus; an extracorporeal immunity absorbingapparatus that contacts between separated plasma and the therapeuticbase material for cardiac disease described in the above 7 and 8; and areflux apparatus which mixes plasma treated with the above describedextracorporeal immunity absorbing apparatus into separated hemocytes,and send it back into body again.

11. The apparatus for therapy of cardiac disease according to the above10, wherein the cardiac disease is a dilated cardiomyopathy.

12. A method for making an animal model for evaluating cardiac disease,which comprises administering the anti-cardiac troponin I antibody.

13. The method for making an animal model for evaluation of cardiacdisease according to the above 12, wherein the cardiac disease isdilated cardiomyopathy.

14. A method of screening a substance for therapy of cardiac disease,which comprises determining the effect of a test substance to cardiacdisease after administration of the substance to an animal made by themethod according to the above 12.

15. A method of screening a substance for therapy of dilatedcardiomyopathy, which comprises determining the effect of a testsubstance to cardiac disease after administration of the substance to ananimal made by the method according to the above 13.

16. A pharmaceutical composition for therapy of dilated cardiomyopathy,which comprises a substance that inhibits the production of anti-cardiactroponin I autoantibody as an active ingredient.

17. A diagnosis method of dilated cardiomyopathy, which comprisesmeasuring an amount of anti-cardiac troponin I autoantibody.

In the present invention, cardiac troponin I protein includes cardiactroponin I of mammal, such as mouse, rat, hamster, guinea pig, dog, pig,monkey and human.

Human cardiac troponin I protein includes the 210-aa protein that hasthe sequence indicated protein ID: CAA38102.1 or a mutant protein thathas at least one or more than two amino acid substitution thereof.Moreover, human cardiac troponin I protein and its mutant proteininclude the protein that has at least 70%, homology, preferably 80 or90% homology, more preferably 95% homology in the consecutive amino acidregion of which length is at least 10 aa, preferably at least 30, 40,50, 60, 100, or 200 aa.

A partial protein of cardiac troponin I includes the partial protein ofcardiac troponin I protein and its mutant protein. These partialproteins can bind the anti-cardiac troponin I autoantibody and containat least continuous 10 aa, preferably at least continuous 30, 40, 50,60, 100 or 200 aa of normal or mutational cardiac troponin I. Moreover,in case of human, more preferably the partial protein has the regionincluding less than 30% sequence homology to slow skeletal muscle typetroponin I (RefSeq data sources; protein ID: NP_(—)003281), and quickskeletal muscle type troponin I (RefSeq data sources; protein ID:BC032148). Furthermore, partial protein of cardiac troponin I may befused with another protein.

In the present invention, anti-cardiac troponin I antibodies include anyantibodies which can specifically bind to cardiac troponin I and partialprotein thereof and anti-cardiac troponin I autoantibody. Polyclonal ormonoclonal antibodies of the present antibody may be obtained fromimmunized rodents by using a general method (Kohler, Milstein, Nature,1975, 256, p 495-497). The antibodies and its partial fragment are anyantibodies such as polyclonal antibodies, monoclonal antibodies, andpartially or fully humanized antibodies and its shortened type (such asF(ab′)2, Fab′, Fab, or F(v)). Fragments of F(ab′)2, Fab′, Fab, or F(v)antibody can be obtained by cleavages of complete antibody withproteases, if necessary additively with reduction. By isolating cDNAfrom the antibody-producing hybridoma, and using the expression vectormade by genetical modification, the antibody, its fragment and the fusedprotein of the antibody fragment with another protein can be obtained.

An anti-cardiac troponin I autoantibody means the antibody that isproduced by autoimmunocyte and that reacts with autologous cardiactroponin I protein. Autoantibodies can be comprised of any subtype ofthe immunoglobulin. An anti-cardiac troponin I antibody may be obtainedin the form contained in circulated blood, collected blood, sera, bloodplasma, urine and tissue or substantial pure state purified therefrom.The autoanitibody may be obtained by isolating or purifying from theobjective patients' blood. Furthermore, anti-cardiac troponin Iautoantibody and segment thereof or fusion protein between segmentthereof and another protein may be produced by using geneticallymodified expression vector. The vector may be obtained by using isolatedautoantibody cDNA from substantial single lymphocyte of objectivepatients which produces anti-cardiac troponin I autoantibody.

In the present invention, the target tissue includes a part of thetissue such as heart, atrium, ventricle, valve, heart wall, inferiorvena cava, superior vena cava, aorta, pulmonary trunk, left superiorpulmonary vein, coronary artery, and coronary vein and primary culturedcell and cell line derived from the above tissues. Preferably,cardiomyocytes and the tissue expressing cardiac troponin I ortissue-derived cells which express cardiac troponin I.

In the present invention, test substance is selected from peptide,polypeptide, nucleotide, polynucleotide, polysaccharide, naturalproduct, fermentation product, cell extract, plant extract, animaltissue extract, antibody, antiserum, derivatives thereof, modifiedsubstance thereof, synthesized compound, organic synthesized compoundand the like.

A substance that inhibits the effect of autoantibodies on the targettissue may be every substance that inhibits the effect of autoantibodiesexerted by binding to the tissue which expresses or contains theirantigens, preferably antigenic protein, a partial protein thereof, andmodified protein thereof, more preferably the base material coupled withantigenic protein, a partial protein thereof and modified proteinthereof.

A substance that inhibits interaction between anti-cardiac troponin Iantibody and cardiac troponin I may be every substance that inhibits thebinding of cardiac troponin I to cardiac troponin I antibody. Oneexample is a substance that binds to a part responsible for antigenrecognition. Preferable substance is such as cardiac troponin I protein,a portion thereof and modified protein thereof, and more preferable isthe base material coupled with cardiac troponin I protein, a partialprotein thereof and modified protein thereof.

A substance of the present invention that inhibits the effect ofanti-cardiac troponin I to the target tissue may be every substance thatinhibits binding of antibody to the target tissue, the enhancement orsuppression of electrophysiological signal that occurs by the effect ofthe antibody, and tissue damage by the effect of the antibody. Oneexample is a substance that binds to antigen-recognition site of theantibody. Cardiac troponin I or its partial protein, or modified proteinthereof is preferable. A base material coupled with cardiac troponin Ior its partial protein or modified protein thereof is more preferable.

A screening method of the substance that inhibits interaction betweenanti-cardiac troponin I antibody and cardiac troponin I, may be done inthe way to evaluate interaction between anti-cardiac troponin I antibodyand cardiac troponin I qualitatively and quantitatively. The screeningmay be carried out by such as radioimmunoassay (RIA), chemiluminescentimmunoassay (CIA), enzyme immunoassay (EIA), Western blotting, orBiacore. An objective substance can be selected by calculation of thestrength of the interaction between anti-cardiac troponin I antibody andcardiac troponin I under existence of control or test substance.

A screening of the substance that inhibits the effect of anti-cardiactroponin I antibody on the target tissue can be carried out byevaluating the effect to target tissue qualitatively and quantitatively.In case that target tissue is a certain type of cells, it can be carriedout by the measurement of viable cells (such as MTT assay, XTT assay andFACS), of activity in cell proliferation, of cell activity (such as ATPsynthesis activity), of cell damage activity (such as LDH releaseactivity) and the like. In case that cardiomyocyte is chosen forexperimental substance, it can be carried out by evaluating the effectof test substance on the increase of Ca²⁺ influx current under existenceor non-existence of anti-cardiac troponin I antibodies. In every method,an objective substance can be selected by the comparison of Ca²⁺ influxcurrent between when control substance is added into the cell expressingcardiac troponin I together with anti-cardiac troponin I and when testsubstance is added into the cell expressing cardiac troponin I togetherwith anti-cardiac troponin I. In case that the target tissue is raw,tissue injury can be evaluated by staining or immunostaining.

The substance selected by the screening for a substance that inhibitsinteraction between anti-cardiac troponin I antibody and cardiactroponin I or that inhibits the effect of anti-cardiac troponin I on thetarget tissue may be produced or synthesize substantially in a pure formas a selected peptide, polypeptide, nucleotide, polynucleotide,polysaccharide, natural product, fermentation product, cell extract,plant extract, animal tissue extract, antibody, antiserum, derivativesthereof, modified substance thereof, synthesized compound, organicallysynthesized compound and the like, by using arbitrary method.

In case that the selected substance is peptide or polypeptide,preferably cardiac troponin I or its partial protein, or modifiedprotein thereof, they may be produced by a protein expression system(host-vector system) with transgenic technology, such as bacteria-,yeast-, insect cell-, mammalian cell-expression system.

A vector may include Escherichia coli plasmid (such as pBR322, pBR325,pUC12, pUC13), Saccharomyces cerevisiae plasmid (such as pSH19, pSH15),bacteriophage such as λ phage, animal virus (such as retrovirus,vaccinia virus, baculovirus), pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neoand the like. A promoter used in the present invention is any one ofpromoters that are both adequate to express gene and suitable for host.For example, in case that animal cell is used as host, SRα promoter,SV40 promoter, LTR promoter, CMV promoter and HSV promoter may be used.Among these, CMV (cytomegalovirus) promoter and SRα promoter arepreferable. In case that host is insect cell, polyhedrin promoter andP10 promoter are preferable as promoter.

The objective peptide may be obtained by Escherichia genera which istransformed with an expression vector and cultured in an adequatemedium. If bacteria's signal peptide (such as pelB signal peptide) maybe used, the objective peptide may be secreted in its periplasm.Furthermore, a fusion protein of the peptide with the other peptide maybe produced.

In case that the objective protein may be expressed in mammalian cell,for example, to secrete the objective peptide in the culture medium,adequate host mammalian cell may be transformed with an adequateexpression vector which contained cDNA coding the objective protein, andthen the transformant is cultured in an adequate medium.

A host may include, such as Escherichia species, Bacillus species, yeastcell, insect cell, insect, animal cell, and the like. Examples ofEscherichia species include Escherichia coli K12, DH1, JM103, HB101,JM109, DH5, DH5α and the like. In case that virus is AcNPV, cell linederived from larval of Spodoptera frugiperda (Sf cell) may be chosen asa host. In case that virus is BmNPV, such as cell line derived fromsilkworm larva (BmN cell) may be chosen as a host. Sf cell includes Sf9cell (ATCC CRL1711), Sf21 (Vaughn, J. L., In Vivo, 1977, Vol. 13, p213-217). Insect includes a larva of silkworm (Bombyx mori larva).Animal cell includes, monkey-derived COS-7 cell line, Vero cell line,Chinese hamster ovary cell line (hereinafter referred to as CHO cell),dhfr gene-deficient Chinese hamster cell line (hereinafter referred toas CHO (dhfr-) cell), mouse L cell, mouse AtT-20, mouse myeloma cell,rat GH3, human FL, HEK 293 cell, etc.

Transformation of Escherichia species can be carried out in accordancewith methods as disclosed in Proceedings of the National Academy ofSciences of the United State of America, Vol. 69, 2110 (1972), Gene,Vol. 17, 107 (1982) and the like. Transformation of yeast cell can becarried out in accordance with methods as disclosed in Methods inEnzymology, 194, 182-187 (1991) and the like. Transformation of insectcells or insects can be carried out in accordance with methods asdisclosed in Bio/Technology, 6, 47-55, (1988) and the like.Transformation of Bacillus species can be carried out in accordance withmethods as disclosed in Molecular & General Genetics, Vol. 168, 111(1979) and the like. Transformation of animal cells can be carried outby methods as disclosed in Cell Engineering, separate vol. 8, New CellEngineering Experiment Protocol, 263-267 (1995) (Shujun Company),Virology, Vol. 52, 456 (1973) and the like.

Cardiac troponin I and partial protein thereof may be produced as afusion protein with a portion of the other protein. Suitable partialproteins for fusion include histidine tag, Fc fragment of immunoglobulinconstant region, GST (glutathione S-transferase) and the like. These canbe beneficial to purification steps after mass production, can increasethe solubility of fusion protein, and can be expected to increase theconformational stability.

A modified protein includes enzymatically or chemically aminated,phosphorylated, acylated, methylated, esterificated, myristylated,disulfidated, ubqutinated, sugar-residues added, oligosaccharide-added,polysaccharide-added, oxidated, reduced, or hydrolyzed protein at thespecific or arbitrary site of the protein.

A protein obtained the above method may be purified in a general method,for example, ion exchange chromatography, gel filtration, hydrophobicchromatography, affinity chromatography (such as, Ni²⁺ ion column,anti-GST antibody affinity column, Protein A affinity column).

A base material coupled with a substance that inhibits the interactionbetween anti-cardiac troponin I antibody and cardiac troponin I or theeffect of the anti-cardiac troponin I antibody on target tissue is amaterial binding the substance electrostatically, chemically, orbiochemically. Among the source of the material, insoluble carrierincludes a synthetic polymer (such as polystyrene, polypropylene,polyamide, polyimide, polyaromatic vinyl compound, polyester, polymethylmethacrylate, polysulfone, polyethylene, polyvinyl alcohol, andpolytetrafluoroethylene), a natural polymer (such as cellulose, agarose,sepharose, dextran, chitin, chitosan) and a derivative thereof. Inaddition, it also includes metals, silica gels, diatomaceous earth,celite, zeolite, porous glasses, ionic exchange resin and a derivativethereof, organic or inorganic porous material, magnetic bead, micro beadand the like. The source of the material may not be limited to the abovedescribed.

A form of base material in the present invention includes particle,fiber, membrane, hollow fiber and the like. A base material form of thepresent invention is not limited, but particle or fiber is preferablebecause of handling convenience. In case of particulate material,particle size is 10-5000 μm in usual, preferably 50-1000 μm at the pointof the clogging in filling up a column and the absorption rate ofadsorbates, but it is not limited to the above described. Furthermore,adsorbates can be sterilized by auto clave sterilization, EOGsterilization, γ-ray irradiating sterilization and the like.

A binding of the substance to the base material can be carried out byamine coupling, coupling by the use of biotin-avidin interaction,thiolate method, amino-coupling method by the use of carboxyl group,cyanogen bromide method and the like.

An animal model for evaluation of cardiac disease, made byadministration of the anti-cardiac troponin I antibody is a mammal thatdevelops cardiac disease like symptom due to damage to cardiac functionor tissue by administration anti-cardiac troponin I antibody once orintermittently more than twice. The preferable animal is mouse, rat,hamster, guinea pig, dog, pig, and monkey.

A method for selection of a therapeutic base material for cardiacdisease by the administration of test substance to the animal affectedwith cardiac disease due to administration of the anti-cardiac troponinI antibody is to evaluate improvement of cardiac dysfunction,histological or morphological damage due to administration of theanti-cardiac troponin I antibody. In case that cardiac disease isequivalent of human dilated cardiomyopathy, end-systolic volume,end-diastolic volume, ejection fraction, pressure driving rate,ventricular end-systolic volume elastance, systemic peripheral vascularresistance index, end-diastolic pressure, right-atrial pressure and thelike may be evaluated. Cardiac tissue damage also may be evaluated bytissue stain or immunological tissue stain. Test substance mixed withpharmacologically allowable solvent may be administrated orally orparenterally, before, during, or after administration of theanti-cardiac troponin I antibody.

A therapy of anti-cardiac troponin I autoantibody-related diseases maybe treated by removing anti-cardiac troponin I autoantibody from apatient's blood and administration of pharmaceutical component fortherapy with substance that inhibits the effect of cardiac troponin Iautoantibody on target tissue or cell, or the production of anti-cardiactroponin I antibody.

Anti-cardiac troponin I autoantibody-related diseases include cardiacdisease, especially dilated cardiomyopathy.

The method of removing anti-cardiac troponin I autoantibody from apatient's blood may be performed by the method consisted the followingstep; (1) the separation of a patient's blood into hemocyte and plasma,(2) the contact between separated plasma and a base material coupledwith a substance that inhibits the interaction between anti-cardiactroponin I antibody and cardiac troponin I, and (3) sending both themixture of treated plasma in step (2) and separated hemocyte back againinto the patient body.

A therapeutic apparatus that removes the anti-cardiac troponin Iautoantibody from a patient's blood is a therapeutic apparatus forcardiac disease, preferably for dilated cardiomyopathy containing thefollowing as essential components; (1) an apparatus for plasmaseparation of collected blood from a patient into plasma and hemocyte,(2) an apparatus for extracorporeal immunity absorption that contactsbetween separated plasma and therapeutic base material coupled with asubstance that inhibits the interaction between anti-cardiac troponin Iantibody and cardiac troponin I, and (3) reflux apparatus for sendingthe treated plasma in step (2) back after mixing with separatedhemocyte.

A pharmaceutical composition for therapy of dilated cardiomyopathycontaining a substance that inhibits the production of anti-cardiactroponin I autoantibody as an effective component contains a substancethat inhibits the production of anti-cardiac troponin I autoantibody. Asubstance that inhibits the production anti-troponin I autoantibody, isany substance chosen from, for example, a substance that directly orindirectly inhibits the antibody production itself or the extarcellularsecretion of the antibody, a substance that inhibits any of thedifferentiation, maturation or proliferation of the clonal lymphocytesproducing autoantibody, or a substance that induces apoptosis of thelymphocytes or injures specifically the lymphocyte. In addition, asubstance that inhibits the production anti-troponin I autoantibody isalso chosen as an immunosuppressive agent that inhibits the entireimmunoreactions that is related to the onset of the said disease.

In the present invention, the measurement of cardiac troponin Iautoantibody may be carried out by radioimmunoassay (RIA),chemiluminescence immunoassay (CIA), enzyme immunoassay, westernblotting, Biacore measurement, and the like. These methods formeasurement may be consist of at least (1) contacting and then reactingblock for measurement which is electrostatically, chemically orbiochemically bound by cardiac troponin I or partial protein thereofwith objective sample, such as blood, serum, blood plasma, urine ortissue extract, (2) adding and then reacting radioactivesubstance-labeled, enzyme-labeled or affinity substance-labeledsecondary antibody, (3) in case of radioactive substance-labeledantibody, measuring the emitting bioluminescence, or coloring signal byscintillation, or in case of enzyme-labeled antibody, measuring theemitting bioluminescence, or coloring signal by adding or reactingchemical luminescent substrate, or chemical coloring substrate. Eachstep may have a washing step arbitrarily.

A diagnosis of anti-cardiac troponin I autoantibody-related diseases maybe carried out by measuring the amount of the antibody in collectedblood, serum, plasma, urine or tissue of the patient in the abovemethod.

A diagnostic reagent of anti-cardiac troponin I autoantibody-relateddiseases may be consist of at least (1) a block for measuring which iselectrostatically, chemically, or biochemically bound by human cardiactroponin I or partial protein thereof, (2) a dilution buffer for blood,serum, plasma, urine, or tissue extract of the patient, (3) radioactivesubstance-labeled, enzyme-labeled, or affinity substance-labeledsecondary antibody, and (4) affinity substance-labeled enzyme in case ofaffinity substance-labeled secondary antibody, (5) in case ofradioactive substance-labeled secondary antibody, scintillation; or incase of affinity substance-labeled or enzyme-labeled secondary antibody,chemical luminescent substrate, or chemical coloring substrate.

INDUSTRIAL APPLICABILITY

Application to Pharmaceuticals:

The substance of the present invention that inhibits the interactionbetween anti-cardiac troponin I antibody and cardiac troponin I, theeffect of anti-cardiac troponin I antibody to the target tissue, or theproduction of anti-cardiac troponin I autoantibody is useful for thetherapy of the anti-cardiac troponin I-derived disease when it isadministrated in pharmaceutically acceptable way. Cardiac diseaseincludes cardiomyopathy (dilated cardiomyopathy, familial hypertrophiccardiomyopathy, hypertrophic cardiomyopathy, primary cardiomyopathy,idiopathic cardiomyopathy, secondary cardiomyopathy, congestivecardiomyopathy, restrictive cardiomyopathy), myocardial infarction,post-myocardial infarction syndrome, post-pericardial incision syndrome,pericarditis (pericarditis due to collagen disease, rheumaticpericarditis, idiopathic pericarditis), endocarditis, myocarditis andthe like.

The substance of the present invention may be used as a solidpreparation or liquid preparation for internal use which is formulatedto mix with pharmacologically acceptable solvent in case of oraladministration, or as a injection, external medicine, suppository andthe like which is formulated to mix with pharmacologically acceptablesolvent in case of parenteral administration.

Examples of the solid preparations for internal use for oraladministration include tablets, pills, capsules, powders, granules andthe like. The capsules include hard capsules and soft capsules.

Such a solid preparation for internal use is prepared by a formulationmethod commonly employed by using one or more than two active substanceseither as the very thing or as a mixture with an excipient (lactose,mannitol, glucose, microcrystalline cellulose, starch, etc.), a binder(hydroxypropylcellulose, polyvinylpyrrolidone, magnesium metasilicatealuminate, etc.), a disintegrating agent (calcium cellulose glycolate,etc.), a lubricant (magnesium stearate, etc.), a stabilizer and adissolution aid (glutamic acid, aspartic acid, etc.). If necessary, itmay be coated with a coating agent (sucrose, gelatin,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.).It may be coated with two or more layers. Moreover, capsules made of anabsorbable substance such as gelatin are involved in the scope thereof.

The liquid preparations for internal use for oral administration includepharmaceutically acceptable aqueous solutions, suspensions, emulsions,syrups, elixirs and the like. Such a liquid preparation is prepared bydissolving, suspending or emulsifying one or more active substances in adiluent commonly employed (purified water, ethanol or a mixture thereof,etc.). Such liquid forms may also further comprise some additives suchas humectants, suspending agents, emulsifying agents, sweetening agents,flavoring agents, aroma, preservatives, buffers and the like.

The injections for parenteral administration include solutions,suspensions, emulsions and solid injections to be dissolved or suspendedbefore use. Such an injection is used by dissolving, suspending oremulsifying one or more active substances in a solvent. The solventincludes, for example, distilled water for injection, physiologicalsaline, vegetable oils, alcohols such as propylene glycol, polyethyleneglycol and ethanol, and mixtures thereof. The injection may furthercontain a stabilizer, a dissolution aid (glutamic acid, aspartic acid,Polysorbate 80 (registered trademark), etc.), a suspending agent, anemulsifier, a soothing agent, a buffer, a preservative, and the like.Such an injection may be produced by sterilizing at the final step oremploying an aseptic process. Alternatively, it is also possible that anaseptic solid product such as a freeze-dried product is produced andsterilized or dissolved in aseptic distilled water for injection oranother solvent before use.

Other formations for parenteral administration include liquidpreparation for external use, ointment, swab preparation, inhalant,spray, suppository and pessary for intravaginal administration which isprescribed in a general method.

Atomized agents, inhalations and sprays may contain, in addition to adiluent commonly employed, a stabilizer such as sodium hydrogen sulfite,a buffering agent for imparting isotonicity, for example, an isotonicagent such as sodium chloride, sodium citrate or citric acid. Methodsfor producing a spray are described in detail in, for example, U.S. Pat.No. 2,868,691 and U.S. Pat. No. 3,095,355.

A pharmaceutical component for therapy of the present invention isusually administrated locally or systemically via an oral or parenteralroute.

The dose of these compounds depends on the age, weight and symptom ofthe patient, the remedial value, the administration method, thetreatment time, etc. In practice, however, these compounds areadministered orally once or several times per day each in an amount of0.1 mg to 100 mg per adult, parenterally once or several times per dayeach in an amount of 0.01 mg to 30 mg, per adult or continuouslyadministered into vein for 1 hour to 24 hours per day.

It goes without saying that the dose of these compounds may be less thanthe aforementioned value or may need to exceed the aforementioned rangebecause the dose varies under various conditions as mentioned above.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 indicates the result of purification and identification of the30-kDa autoantigen; (a) silver-staining (left) and western blotting withPD-1-deficient mouse serum (right), (b) anion-exchange columnchromatography, (c) cation-exchange column chromatography, (d) reversephase HPLC column chromatography, and (e) comparison of the amino acidsequences of cardiac troponin I with purified band.

FIG. 2 indicates immunological character of cardiac troponin I inexample 3; (a) immunoprecipitation of cardiac troponin I from heartextract, (b) competition with recombinant cardiac troponin I, and (c)immunostaining of normal cardiac slice.

FIG. 3 indicates hemodynamic parameters of mice treated withanti-cardiac troponin I antibody in example 4; (a) the graph of thepressure-to-volume relationship of control IgG administrated mice, (b)the graph of the pressure-to-volume relationship of anti-cardiactroponin I administrated mice (dotted line indicates end-systolic andend-diastolic pressure-to-volume relationship), (c) left-ventricularpressure curve of control IgG treated mice (left) and of anti-cardiactroponin I antibody treated mice (right), (d) the gradient of inductionperiod of systolic pressure change per unit time in control Ig treatedmice (left) and in anti-cardiac troponin I antibody treated mice(right), (e) comparison between control IgG treated mice andanti-cardiac troponin I antibody treated mice in ESV (end-systolicvolume), EDV (end-diastolic volume), EF (ejection fraction) and maximumpressure driving rate (dP/dt_(max)).

FIG. 4 indicates electrophysiological analysis using cardiomyocytesderived from dilated hearts of PD-1-deficient mice and effect ofanti-cardiac troponin I antibody on normal cardiomyocytes; depolarizedCa²⁺ current (lower panel) evoked by a test pulse (upper panel) in (a)wild type (wt) mice and in (c) PD-1-deficient mice; voltage-currentrelationships of (b) wt mice and of (d) PD-1-deficient mice; (e)-(i) theeffect of antibodies against cardiac troponin I on the Ca²⁺ current ofnormal ventricular cardiomyocytes

FIG. 5 indicates data of improvement in hemodynamics of anti-cardiactroponin I antibody treated mice.

BEST MODE FOR CARRYING OUT THE INVENTION

The following reference examples and examples illustrate, but do notlimit the present invention.

Example 1

Purification and Identification of the 30-kDa Autoantigen:

(a) Solubilization and Purification of 30-kDa Band

The 30-kDa antigen was purified from extract of rat hearts. Rat heartswere homogenized in a detergent-free buffer (0.25 M sucrose withcomplete protease inhibitor cocktail (Roche)), dialyzed againstBis-Tris, buffer (20 mM Bis-Tris, 50 mM NaCl; pH 6.8) and fractionatedby a mono-Q column (Amersham Biosciences) with the NaCl gradient. Eachfraction was detected by western blotting with sera of PD-1-deficientmice. In FIG. 1 (a), detergent-containing (lane 1) and detergent-free(lane 2) rat heart extracts, fraction 22 of Q sepharose column (lane 3)and fraction 33 of S sepharose column (lane 4) were silver-stained(left) and western blotted (right) with PD-1-deficient mouse serum.

(b) Anion-Exchange Column Chromatography

Rat heart extracts were fractionated by a mono-Q column (AmershamBiosiences) with the NaCl gradient and each fraction was detected bywestern blotting using PD-1-deficient mouse sera. Antigen-containingfractions (fraction 21 and 22) were indicated by shade in FIG. 1 (b).

(c) Cation-Exchange Column Chromatography

Fractions 21 and 22 of the mono-Q sepharose chromatography were dialyzedagainst MES buffer (50 mM MES, 50 mM NaCl; pH 5.5) and fractionated by amono-Q column (Amersham Biosciences). Antigen-containing fractions(fraction 33 and 34) were indicated by shadow in FIG. 1 (c).

(d) Reverse Phase HPLC

Positive fractions were separated with SDS-PAGE. A band approximately 30kDa in size was excised, in-gel-digested with trypsin and separated byreverse phase HPLC (FIG. 1 (d)).

(e) Comparison of the Amino Acid Sequences of Cardiac Troponin I withthat of Purified Band

The amino acid sequence of the peptide that was indicated by an arrow inFigure (d) was identified and compared with that of rat, mouse, humanand caw cardiac troponin I. The obtained sequence was same as that ofcardiac troponin I (cTnI) (FIG. 1 (e)).

Example 2

Generation of a Monoclonal Antibody Against Cardiac Troponin I:

Complementary DNA of mouse cardiac troponin I was cloned from mousecardiac mRNA using reverse transcriptase and PCR. A GST-fusionrecombinant protein containing cardiac troponin I was made. Mousecardiac troponin I protein was obtained by subsequent PreScissionprotease cleavage (Amersham Pharmacia) of the fusion protein.Immunization of PD-1-deficient mice with this recombinant cardiactroponin I (>95% pure as judged by SDS-PAGE) allowed us to establishmonoclonal antibody against mouse cardiac troponin I (the antibodyproduced by hybridoma designated cTnI-1-4A: it was deposited Apr. 2,2003 at the International Patent Organism Depositary of NationalInstitute of Advanced Industrial Science and Technology (Tsukuba Central6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan; deposit number: FERMP-19287)) and transfer of jurisdiction to international deposit Mar. 5,2004 (international deposit number: FERM BP-08653)). The monoclonalantibody was detected a 30-kDa band by western blotting of the heartextracts. The size of the band was identical to that recognized byPD-1-deficient mouse serum and commercial goat polyclonal antibody tomouse cardiac troponin I (Santa Cruz).

Example 3

Immunological Characterization of Cardiac Troponin I:

(a) Immunoprecipitation of Cardiac Troponin I from Heart Extract

Crude heart extracts were immunoprecipitated by goat polyclonal antibodyto cardiac troponin I (Santa Cruz). Supernatant (sup) and precipitate(ppt) were obtained. These were checked for the presence of the 30-kDaantibody by western blotting using PD-1-deficient mouse serum. Theresult was indicated in FIG. 2 (a) (in the figure, crude means crudeheart extract, and sup means supernatant, and ppt means precipitation.).The 30-kDa antigen was detected in the precipitate fraction by westernblotting using anti-cardiac troponin I monoclonal antibody. The size ofthe band was same as that identified by PD-1-deficient mouse serum andgoat polyclonal antibody to mouse cardiac troponin I (Santa Cruz).

(b) Competition with Recombinant Cardiac Troponin I

PD-1-deficient mouse serum (mouse #101, #104 and #117) was used forwestern blotting of normal mouse heart extract. For the competitionassay, recombinant cardiac troponin I was added to immune blot solutionat 1, 10, and 100 μg/ml. The 30-kDa antigen recognized by PD-1-deficientmouse serum was depleted by immunoprecipitation with antibodies toanti-myocardial troponin I and was recoverable from the pellet fraction(FIG. 2 (b)).

In addition, the intensity of the band recognized by PD-1-deficientmouse serum was weakened in a dose-dependent manner by the addition ofrecombinant cardiac troponin I, whereas the minor bands were unaffected(FIG. 2 (b)). Thus, the band was confirmed to consist solely of cardiactroponin I.

(c) Immunostaining of Normal Heart Section

The subcellular localization of cardiac troponin I was analyzed byimmunoelectron microscopy. The image of normal heart section stainedwith the 30-kDa autoimmune antibody was shown in FIG. 2 (c).Anti-cardiac troponin I monoclonal antibody stained the surface ofcardiomyocytes of normal heart. Arrows indicate signals on the surfaceof T-tubes, arrowheads indicate signals on the surface of cytomembrane,MT means mitochondria and T means T-tubule. These signals are detectedon the outer surface of T-tubules, where L type Ca²⁺ channels arecondensed and bound to ryanodine receptor in sarcoplasmic reticulum.

Example 4

Hemodynamics of Mice Treated with Anti-Cardiac Troponin I Antibody:

To examine the left-ventricular pressure-to-volume relationships inanti-cardiac troponin I antibody administrated wild type mice, theventricular function was evaluated by cardiac catheterization.Four-week-old female BALB/c mice were intraperitoneally injected with600 μg of monoclonal antibodies or 600 μg of control mouse IgG on days0, 7, 21, 35, 49, 63 and 77 and analyzed on day 82.

FIG. 3 showed the pressure-to-volume relationship in control IgG (FIG.3(a)) or anti-cardiac troponin I administrated mice (FIG. 3(b)); dottedlines indicate end-systolic and end-diastolic pressure-to-volumerelationship), left-ventricular pressure treated with control IgG (FIG.3(c), left) or cardiac troponin I-specific antibodies (FIG. 3(c),right), the gradient of pressure change per unit time (FIG. 3 (d)) incontrol IgG treated mice (left) and anti-cardiac troponin I antibodiestreated mice (right), and comparison of ESV (end-systolic volume), EDV(end-diastolic volume), EF (ejection fraction) and pressure driving rate(FIG. 3 (e); each value shown represents mean±s.e.m.).

Twelve weeks after the initiation of treatments, the left ventricles ofthe antibody-treated mice had increased end-systolic volume (ESV) andend-diastolic volume (EDV) (+198%, and +29%, respectively), whichresulted in the reduction of ejection fraction from 79.3% in controls to50.8% (FIGS. 3 (a), (b), and (e)). Systolic function was markedlydepressed, as reflected by the decreases in pressure development(dP/dt_(max); −35%), the prolongation of the monoexponetial timeconstant of relaxation (t; 10.1 to 11.7 ms) (FIG. 3 (b), (d), and FIG.5). Increases in end-diastolic pressure and right atrium pressure(average right atrium pressure) are representative of the heart failure(FIG. 5). These hemodynamic data indicate that administration ofantibody to cardiac troponin I caused increases in ESV and EDV of theleft ventricle by weakening systolic function.

Example 5

Electrophysiological Analyses in Cardiomyocytes Derived from DilatedHearts of PD-1-Deficient Mice and Effect of Anti-Cardiac Troponin IAntibody on Normal Cardiomyocytes:

The electrophysiological changes in cardiomyocytes of dilated heart ofPD-1-deficient mice were analyzed in order to investigate the in vitroeffects of anti-cardiac troponin I antibodies on isolatedcardiomyocytes. The method of isolation is as follows. Under anesthesia,mice were intubated intratracheally and ventilated. A cannula wasinserted into the ascending part of aorta, and then only heart was takenout and suspended by a cannula. Collagenase was injected via a cannulaand refluxed the entire heart through coronary artery for 30 minutes.After refluxing collagenase-free solution for several minutes, the heartwas put into the beaker and divided gently by tweezers to isolatecardiomyocytes.

Cardiomyocytes were isolated from wild type mice and dilatedcardiomyopathy and each single myocytes were investigated by patchclamp. By replacing extracellular potassium ion by cesium ion, onlycalcium current was measured.

Voltage-dependent Ca²⁺ current (lower panel) evoked by a test pulse(upper panel) in cardiomyocytes from wild type (wt) mice (FIG. 4(a)) andPD-1-deficient mice (FIG. 4 (c)) was plotted and voltage-currentrelationships were shown for wt mice (FIG. 4 (b)) and PD-1-deficientmice (FIG. 4 (d)) (Ca²⁺ current was normalized based on each membranecapacity and plotted as average±S.E.; ● indicates peak value (peak) andΔ indicates plateau value of Ca²⁺ current (plateau).). FIG. 4 (e)-(i)showed the effects of antibodies against cardiac troponin I on the Ca²⁺current of normal ventricular cardiomyocytes. Normal ventricularcardiomyocytes were microperfused with (e) control goat IgG, (f)polyclonal antibodies against cardiac troponin I, and monoclonalantibodies against cardiac troponin I (1-1A, 1-4A, or 3-18A, (g)-(i))and monitored for the inward Ca²⁺ current, evoked by a depolarizingpulse to ±0 mV, every 6 seconds. Each value is presented as ratioagainst the initial magnitude of the Ca²⁺ current ((e)-(i)).

The maximal voltage-dependent Ca²⁺ current was increased about threefoldin cardiomyocytes of dilated hearts (FIG. 4 (a) and (c)). The membranecapacitance of cardiomyocytes didn't change significantly (147±31 pF and169±27 pF in cardiomyocytes from wt and PD-1 deficient mice,respectively). The variance in the Ca²⁺ current didn't change bynormalizing the value of the Ca²⁺ current to membrane capacitance(pA/pF) (FIG. 4 (b), (d)). Therefore, the augmentation of the Ca²⁺current is likely to be attributable to changes in the L-type Ca²⁺channel itself.

In the analysis of the effect of antibodies to cardiac troponin I on theCa²⁺ current of wild-type cardiomyocytes by pouring antibodies tocardiac troponin I from a thin tube, the Ca²⁺ current evoked by thedepolarizing pulse to ±0 mV was plotted every 6 seconds and expressed asa ratio against the initial value, because the depolarizing pulse to ±0mV gave a maximal Ca²⁺ current for the wild-type cardiomyocytes (FIG.4(b)). Addition of the commercial polyclonal antibody and the threemonoclonal antibodies (FIG. 4(g)-(i)) to cardiac troponin I augmentedthe Ca²⁺ current of ventricular and atrial cardiomyocytes by as much as1.5-fold (FIG. 4). Control goat IgG or mouse IgG did not affect the Ca²⁺current over the course of 15 min (FIG. 4). Cardiac troponin I expressedon the surface of cardiomyocytes may be involved, in an unknowncapacity, in regulating the magnitude of the Ca²⁺ current. Takentogether, these data imply that antibodies to cardiac troponin I induceDCM in PD-1-deficient mice by chronically enhancing the Ca²⁺ current incardiomyocytes.

1. A screening method of a substance that inhibits interaction betweenanti-troponin I antibody and cardiac troponin I, which comprisesmeasuring and evaluating the inhibitory activity of a test substance oninteraction between anti-cardiac troponin I antibody and cardiactroponin I after contacting the anti-cardiac troponin I antibody, thecardiac troponin I and the test substance.
 2. A screening method of asubstance that inhibits the effect of anti-troponin I to a targettissue, which comprises measuring and evaluating the inhibitory activityof a test substance on the effect of anti-cardiac troponin I antibody ona target tissue after contacting the anti-cardiac troponin I, the targettissue and the test substance.
 3. The screening method according toclaim 2, wherein the target tissue is a myocardial cell.
 4. Apharmaceutical composition of therapy for cardiac disease which is madeby steps comprising a step of selecting a substance with activity by thescreening method according to any one of claims 1 to 3, a step ofmanufacturing the above selected substance, and a step of mixing thesubstance manufactured by the above described step with apharmaceutically acceptable solvent for formulation.
 5. A method fortreating cardiac disease, which comprises administering to a subject inneed thereof an effective amount of at least one selected from cardiactroponin I protein, a partial protein thereof, and a modified proteinthereof.
 6. The pharmaceutical composition of therapy for cardiacdisease according to claim 4, wherein the cardiac disease is dilatedcardiomyopathy.
 7. A therapeutic base material for cardiac diseasecoupled with a substance selected by the method according to any one ofclaims 1 to 3 or at least one selected from cardiac troponin I, itspartial protein, and a modified protein thereof.
 8. (canceled)
 9. Thetherapeutic base material for cardiac disease according to claim 7,wherein the cardiac disease is dilated cardiomyopathy.
 10. An apparatusfor therapy of cardiac disease, which comprises a plasma separatingapparatus; an extracorporeal immunity absorbing apparatus that contactsbetween separated plasma and the therapeutic base material for cardiacdisease according to claim 7; and a reflux apparatus which mixes plasmatreated with the above described extracorporeal immunity absorbingapparatus into separated hemocytes, and send it back into body again.11. The apparatus for therapy of cardiac disease according to claim 10,wherein the cardiac disease is a dilated cardiomyopathy.
 12. A methodfor making an animal model for evaluating cardiac disease, whichcomprises administering the anti-cardiac troponin I antibody.
 13. Themethod for making an animal model for evaluation of cardiac diseaseaccording to claim 12, wherein the cardiac disease is dilatedcardiomyopathy.
 14. A method of screening a substance for therapy ofcardiac disease, which comprises determining the effect of a testsubstance to cardiac disease after administration of the substance to ananimal made by the method according to claim
 12. 15. A method ofscreening a substance for therapy of dilated cardiomyopathy, whichcomprises determining the effect of a test substance to cardiac diseaseafter administration of the substance to an animal made by the methodaccording to claim
 13. 16. A method for treating dilated cardiomyopathy,which comprises administering to a subject in need thereof a substancethat inhibits the production of anti-cardiac troponin I autoantibody.17. A diagnosis method of dilated cardiomyopathy, which comprisesmeasuring an amount of anti-cardiac troponin I autoantibody.
 18. Amethod for treating for cardiac disease, which comprises administeringto a subject in need thereof an effective amount of the pharmaceuticalcomposition according to claim
 4. 19. The method according to claim 5 or18, wherein the cardiac disease is dilated cardiomyopathy.